The RNA helicase DHX33 has been shown to be a critical regulator of cell proliferation and growth. However, the underlying mechanisms behind DHX33 function remain incompletely understood. We present original evidence in multiple cell lines that DHX33 transcriptionally controls the expression of genes involved in the cell cycle, notably cyclin, E2F1, cell division cycle (CDC), and minichromosome maintenance (MCM) genes. DHX33 physically associates with the promoters of these genes and controls the loading of active RNA polymerase II onto these promoters. DHX33 deficiency abrogates cell cycle progression and DNA replication and leads to cell apoptosis. In zebrafish, CRISPR-mediated knockout of DHX33 results in downregulation of cyclin A2, cyclin B2, cyclin D1, cyclin E2, cdc6, cdc20, E2F1, and MCM complexes in DHX33 knockout embryos. Additionally, we found the overexpression of DHX33 in a subset of non-small-cell lung cancers and in Ras-mutated human lung cancer cell lines. Forced reduction of DHX33 in these cancer cells abolished tumor formation in vivo. Our study demonstrates for the first time that DHX33 acts as a direct transcriptional regulator to promote cell cycle progression and plays an important role in driving cell proliferation during both embryo development and tumorigenesis.

b DEAD/DEAH box RNA helicases play essential roles in numerous RNA metabolic processes, such as mRNA translation, premRNA splicing, ribosome biogenesis, and double-stranded RNA sensing. Herein we show that a recently characterized DEAD/ DEAH box RNA helicase, DHX33, promotes mRNA translation initiation. We isolated intact DHX33 protein/RNA complexes in cells and identified several ribosomal proteins, translation factors, and mRNAs. Reduction of DHX33 protein levels markedly reduced polyribosome formation and caused the global inhibition of mRNA translation that was rescued with wild-type DHX33 but not helicase-defective DHX33. Moreover, we observed an accumulation of mRNA complexes with the 80S ribosome in the absence of functional DHX33, consistent with a stalling in initiation, and DHX33 more preferentially promoted structured mRNA translation. We conclude that DHX33 functions to promote elongation-competent 80S ribosome assembly at the late stage of mRNA translation initiation. Our results reveal a newly recognized function of DHX33 in mRNA translation initiation, further solidifying its central role in promoting cell growth and proliferation. Mammalian cells maintain tight control of global mRNA translation through the production of ribosomes (1, 2); deregulation in mRNA translation is frequently found in human diseases (3-6) and is regarded as one of the many factors contributing to cancer development (7-9).Most eukaryotic protein translation initiation occurs by an ordered assembly of a preinitiation complex on the 5= cap of mRNA (10). After mature mRNA is transported into the cytosol, the distinct 5= cap of mRNA is recognized and bound by a large protein complex comprising eukaryotic initiation factor 4E (eIF4E), eIF4A, and eIF4G as well as poly(A)-binding protein (PABP) (1,11,12). These factors coordinately prevent mRNA degradation while priming mRNAs for translation initiation.The initial step in mRNA translation involves formation of a ternary complex between eIF2-GTP, Met-tRNA interference, and small 40S ribosomal subunits. This process is stimulated by the translation initiation factors eIF1, eIF3, eIF4F, and eIF5 (13). This large complex, termed the 43S preinitiation complex, attaches to the activated 5= cap of mRNA. Bound RNA helicases are responsible for unwinding various secondary structures in mRNA as the complex scans along the mRNA from the 5= end to the 3= end until it finds the initiation codon. The 60S large ribosome subunit then joins with the 40S subunit to form an 80S ribosome under guidance from eIF5B-GTP (2, 13). eIF2-GTP and eIF5B-GTP are then hydrolyzed into their GDP forms to promote the assembly of the functional initiation complex (14). The detailed mechanism of how elongation-competent 80S ribosomes are assembled prior to initiation or what triggers initiation is not well understood.Mammalian mRNAs often contain highly structured untranslated regions (UTRs) at the 5= ends of their open reading frame sequences that must be unwound to allow ribosome recruitment and scanning. Not su...

Oncogene c-Myc is frequently amplified and activated in human cancers. Deregulation of c-Myc protein has been shown to occur in 30% of all human cancers, especially in hematopoietic malignancies. As a transcription factor, c-Myc has been shown to regulate up to 15% of all human genome genes, controlling diverse cellular activities including cell cycle, ribosome biogenesis, protein synthesis, metabolism, apoptosis and angiogenesis. In this report, we provide evidence that the RNA helicase DHX33 is a critical downstream target of c-Myc. Myc binds to DHX33 upstream promoter region and stimulates its transcription. Elevated DHX33 protein is pivotal for c-Myc to drive tumor formation. Knockdown of DHX33 to basal levels in c-Myc overexpressing cells significantly reduced cell proliferation, cell migration and anchorage-independent cell growth in vitro and in vivo. Additionally, we found that DHX33 promotes MMP9, MMP14 and urokinase-type plasminogen activator (PLAU) transcription by directly binding to their promoters, thus promoting cancer cell migration. DHX33 protein was overexpressed in a certain subset of human non-Hodgkin's lymphoma tissues. Finally, knockdown of DHX33 significantly inhibits the development of Myc-induced acute myeloid leukemia. Overall, our results implicate the important role for DHX33 in Myc-induced cancer and point toward its potential therapeutic value in Myc driven cancers.

c RNA polymerase I-mediated rRNA production is a key determinant of cell growth. Despite extensive studies, the signaling pathways that control RNA polymerase I-mediated rRNA production are not well understood. Here we provide original evidence showing that RNA polymerase I transcriptional activity is tightly controlled by integrin signaling. Furthermore, we show that a signaling axis consisting of focal adhesion kinase (FAK), Src, phosphatidylinositol 3-kinase (PI3K), Akt, and mTOR mediates the effect of integrin signaling on rRNA transcription. Additionally, we show that in kindlin-2 knockout mouse embryonic fibroblasts, overactivation of Ras, Akt, and Src can successfully rescue the defective RNA polymerase I activity induced by the loss of kindlin-2. Finally, through experiments with inhibitors of FAK, Src, and PI3K and rescue experiments in MEFs, we found that the FAK/Src/PI3K/Akt signaling pathway to control rRNA transcription is linear. Collectively, these studies reveal, for the first time, a pivotal role of integrin signaling in regulation of RNA polymerase I transcriptional activity and shed light on the downstream signaling axis that participates in regulation of this key aspect of cell growth. RNA polymerase I (Pol I) plays a central role in regulating cellular growth and proliferation (1). Eukaryotic cells contain hundreds of ribosomal DNA (rDNA) copies that occupy several different chromosomal locations (2). The production of rRNA can be divided into several steps, i.e., rRNA transcription, modification, and processing, all of which occur in the nucleolus (3, 4). The rate-limiting step is rRNA transcription (1, 5). On sensing of outside stimuli, a preinitiation complex comprised of the transcriptional factors upstream binding factor (UBF), SL1, TBP, Rrn3, and TTF assembles in the promoter region of rDNA. This complex then recruits RNA polymerase I to rDNA loci, and rRNA transcription starts (6-8). In mammalian cells, a single precursor rRNA transcript, 47S rRNA (14.3 kb), is transcribed from rDNA by the RNA polymerase I complex. This large polycistronic transcript encompasses 18S, 5.8S, and 28S rRNAs and includes several spacer regions, which are later processed into distinct rRNA species before assembly into preribosomal subunits (9).The transcriptional activity of Pol I is a fundamental determinant of cell proliferation capacity (3). In rapidly proliferating cells, rRNA production takes more than 50% of all nuclear transcriptional activity. In yeast cells, this percentage can reach more than 80% (10). As such, the tremendous energy consumption demands tight control.At the tissue level, cells attach to the extracellular matrix (ECM) through cell surface receptors termed integrins (11). Integrins are heterodimeric transmembrane receptors comprised of ␣ subunits and ␤ subunits that bind to extracellular ligands, such as laminin, collagen, vitronectin, and fibronectin. Different combinations of the 18 ␣ subunits and 8 ␤ subunits confer specificity on the integrin-ECM interactions (12). After binding ...

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DEAD box proteins are multifunctional proteins involved in every aspect in RNA metabolism and have essential roles in many cellular activities. Despite their importance, many DEAD box proteins remain uncharacterized. In this report, we found DDX59 overexpressed in lung adenocarcinoma. DDX59 knockdown reduced cell proliferation, anchorage-independent cell growth, and caused reduction of tumor formation in immunocompromised mice. In multiple lung cancer cells, we found that DDX59 knockdown inhibits DNA synthesis; wild-type DDX59 but not helicase-defective mutant of DDX59 enhances DNA synthesis. DDX59 knockdown caused reduction of MCM protein levels, decreased the loading of MCM ring protein onto chromatin, and therefore inhibited DNA replication. Our study reveals for the first time that DDX59 has an important role in lung cancer development through promoting DNA replication.

RNA helicase DHX33 was found to regulate the transcription of multiple genes involved in cancer development. But the underlying molecular mechanism remains unclear. Here, we found DHX33 associated extensively with gene promoters at CG-rich region. Its deficiency reduced the loading of active RNA polymerase II at gene promoters. Furthermore, we observed a functional interaction between DHX33, AP-2β, and DNA demethylation protein Gadd45a (growth arrest and DNA damage inductile protein 45a) at specific gene promoters. DHX33 is required to recruit GADD45a, thereby causing local DNA demethylation through further recruiting ten-eleven-translocation (Tet) methylcytosine dioxygenase enzyme, as manifested by reduced 5-hydroxymethyl cytosine levels for a subset of genes after DHX33 deficiency. This process might involve R-loop formation in GC skew as a guidance signal at promoter sites. Our report provides for the first time, to our knowledge, original evidence that DHX33 alters epigenetic marks and regulates specific gene transcription through interaction with Gadd45a.

Proto-oncogene Ras encodes a small GTPase of approximately 20 kDa, which can be tethered to plasma membrane via farnesylation. 1 Upon extracellular signal stimulation, Ras-GDP is activated by Guanine Exchange Factor (GEF) into GTP binding form, which then activates multiple downstream signaling pathways in order to regulate cell growth, proliferation, survival, and differentiation. 2 Three major signaling pathways including PI3K/Akt, Raf/MAP, and Rho GTPase have been found to be activated by Ras as its downstream effectors. 3 Activated Ras can be turned off by GTP hydrolysis into the Ras-GDP form. However, in human cancers, this is frequently disrupted by mutations on Ras. Ras mutation most frequently occurs on G12, which can be mutated into Val, Cys, Ser, or Asp with different frequency in human cancers. 4 The mutation leads to the locking of Ras in the GTP binding form. Although Ras has been found to be frequently mutated in human cancers for a long time, no effective drugs have been discovered and applied in clinics yet. Multiple downstream effectors can be activated